When messenger RNA from ACTH and endorphin secreting mouse pituitary tumor cells (AtT-20-D16v line) is translated in a reticulocyte cell-free protein synthesizing system a protein of apparent molecular weight 28,500 (28.5K ACTH-endorphin) is produced containing the sequences of both adrenocorticotropin (ACTH) and beta-endorphin. Beta-endorphin is located at the C-terminus of the precursor and ACTH is in the middle of the molecule leaving a stretch of 90-100 amino acids at the N-terminus unaccounted for by any known hormone. After labeling AtT-20 cell cultures with radioactive amino acids or sugars, components of processing were isolated by immunoprecipitation (with antisera to ACTH, beta-endorphin, beta-MSH, or the N-terminal region of the precursor), fractionated by SDS gel electrophoresis, and characterized by mapping tryptic peptides and glycopeptides. An initial event in processing in AtT-20 cells is addition of a carbohydrate (CHO) side chain to the N-terminal region of the precursor to form 29K ACTH-endorphin. A second CHO side chain is then added to the ACTH region of the 29K molecule to form 32K ACTH-endorphin or to the N-terminal region to form 34K ACTH-endorphin. Each precursor form is then cleaved to beta-lipotropin and a different intermediate form of ACTH. One ACTH intermediate is converted to alpha(1-39)ACTH and another to the glycosylated form of alpha(1-39)ACTH. N-terminal glycopeptides are also released during this cleavage step. Processing in anterior pituitary cultures of mouse is very similar to that observed in AtT-20 cultures.